Figure 3

Gefitinib inhibits ionizing radiation (IR)-induced ATM-Chk2 activation. A, Western blot analyses for ATM and Chk2 phosphorylation. Cells were pretreated with gefitinib or vehicle (DMSO) for 48 h, exposed to 6 Gy IR, and were harvested and lysed for western blot analysis after 30 min. Blots representative of three independent experiments. B, Cells were harvested, lysed and immunoprecipitated with ATM antibody followed by in vitro kinase assays using GST-Chk1 as a substrate. The amount of phosphor-Chk1 was assessed by immunoblotting using phospho-ser345-Chk1 antibody. C, Cells were grown on coverslips, pretreated with gefitinib for 48 h, and then exposed to 6 Gy IR. After 30 min, cells were fixed and stained with anti-pATM and anti-pChk2 antibodies. Costaining with DAPI was done to show the nuclei. pATM (green), pChk2 (red) and DAPI (blue).